Project Summary/Abstract Human exposure to tropospheric ozone (O3) is a significant public health concern, with millions of Americans living in areas that exceed the National Ambient Air Quality Standard. Acute exposure to O3 causes pulmonary functional decrements and airway inflammation, resulting in significant morbidity in exposed populations. While O3 is a strong oxidizer known to react with a variety of biological molecules, the initiating mechanisms by which it induces inflammation remain unclear. Carbon double bonds in polyunsaturated fatty acids (PUFAs) have widely been recognized as primary targets for O3 attack, leading to formation of free radicals and lipid hydroperoxides. The reactivity of these intermediates with cellular targets is hypothesized to activate pro- inflammatory signaling in human airway epithelial cells (HAEC) exposed to O3. However, dietary supplementation with two omega-3 PUFAs, eicosapentaenoic (EPA) and docosahexaenoic (DHA) acid, have been shown to confer protection against the adverse health effects of exposure to oxidative air pollution in human volunteers, potentially through their metabolism to anti-inflammatory resolvins, maresins and protectins (SPM). Thus, a paradox may exist between the anti-inflammatory effects of omega-3 fatty acids and their role as substrates for the formation of inflammatory lipid hydroperoxides through their reactivity with O3. The goal of this project is to elucidate the role of PUFAs in mediating oxidative and inflammatory responses of HAEC to O3. We hypothesize that omega-3 fatty acid supplementation will modulate the responses of HAEC to O3 exposure. To test this hypothesis, we will first determine the role of lipid hydroperoxide formation in changes to cellular oxidative state during O3 exposure. This will be accomplished through the expression of the genetically-encoded fluorogenic sensor roGFP, which reports the intracellular glutathione redox potential (EGSH) in HAEC in real time. We have previously reported that O3 exposure increases the EGSH in roGFP expressing HAEC. We will then determine the effect of HAEC supplementation with EPA and DHA on O3 induced changes in EGSH. To determine the effect of supplementation on O3 induced inflammation, we will measure the production of both pro-inflammatory cytokines IL-8, IL-6, and IL-1?, and anti-inflammatory SPMs in EPA or DHA supplemented HAEC following O3 exposure. Finally, we will determine the effect of O3 on mitochondrial bioenergetics, an important influencer of cellular oxidative state, in supplemented HAEC using extracellular flux analyses. Taken together, these experiments will elucidate the mechanism by which O3 induces oxidative and inflammatory changes in HAEC, and determine the role of omega-3 fatty acids in this relationship. A greater understanding of the mechanisms of O3 induced cellular activation will support regulatory efforts, and test the efficacy of using diets rich in omega-3 fatty acids to mitigate morbidity from ozone exposure.